RESUMO
Field and laboratory investigations revealed phenotypic, target site and metabolic resistance to permethrin in an Anopheles gambiae s.s. (Diptera: Culicidae) population in Bungoma District, a region in western Kenya in which malaria is endemic and rates of ownership of insecticide-treated bednets are high. The sensitivity of individual An. gambiae s.l. females as indicated in assays using World Health Organization (WHO) test kits demonstrated reduced mortality in response to permethrin, deltamethrin and bendiocarb. Estimated time to knock-down of 50% (KDT50 ) of the test population in Centers for Disease Control (CDC) bottle bioassays was significantly lengthened for the three insecticides compared with that in a susceptible control strain. Anopheles arabiensis from all three sites showed higher mortality to all three insecticides in the WHO susceptibility assays compared with the CDC bottle assays, in which they showed less sensitivity and longer KDT50 than the reference strain for permethrin and deltamethrin. Microplate assays revealed elevated activity of ß-esterases and oxidases, but not glutathione-S-transferase, in An. gambiae s.s. survivors exposed to permethrin in bottle bioassays compared with knocked down and unexposed individuals. No An. arabiensis showed elevated enzyme activity. The 1014S kdr allele was fixed in the Bungoma An. gambiae s.s. population and absent from An. arabiensis, whereas the 1014F kdr allele was absent from all samples of both species. Insecticide resistance could compromise vector control in Bungoma and could spread to other areas as coverage with longlasting insecticide-treated bednets increases.
Assuntos
Anopheles/enzimologia , Anopheles/genética , Resistência a Inseticidas , Inseticidas/farmacologia , Piretrinas/farmacologia , Animais , Bioensaio , Esterases/metabolismo , Feminino , Genótipo , Quênia , Masculino , Nitrilas/farmacologia , Permetrina/farmacologia , Fenilcarbamatos/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Especificidade da EspécieRESUMO
Insecticide resistance is an important factor in the effectiveness of Aedes aegypti control and the related spread of dengue. The objectives of this study were to investigate the status of the organochlorine dichlorodiphenyltrichloroethane (DDT) and pyrethroid (permethrin and deltamethrin) resistance in Trinidad and Tobago populations of Ae. aegypti and the underlying biochemical mechanisms. Nine populations of Ae. aegypti larvae from Trinidad and Tobago were assayed to DDT and PYs using the Centers for Disease Control and Prevention (CDC) time-mortality-based bioassay method. A diagnostic dosage (DD) was established for each insecticide using the CAREC reference susceptible Ae. aegypti strain and a resistance threshold (RT), time in which 98-100% mortality was observed in the CAREC strain, was calculated for each insecticide. Mosquitoes which survived the DD and RT were considered as resistant, and the resistance status of each population was categorised based on the WHO criteria with mortality <80% indicative of resistance. Biochemical assays were conducted to determine the activities of α and ß esterases, mixed function oxidases (MFO) and glutathione-S-transferases (GST) enzymes which are involved in resistance of mosquitoes to DDT and PYs. Enzymatic activity levels in each population were compared with those obtained for the CAREC susceptible strain, and significant differences were determined by Kruskal-Wallis and Tukey's non-parametric tests (P<0.05). The established DDs were 0.01 mg l(-1), 0.2 mg l(-1) and 1.0 mg l(-1) for deltamethrin, permethrin and DDT, respectively; and the RTs for deltamethrin, permethrin and DDT were 30, 75 and 120 min, respectively. All Ae. aegypti populations were resistant to DDT (<80% mortality); two strains were incipiently resistant to deltamethrin and three to permethrin (80-98% mortality). Biochemical assays revealed elevated levels of α-esterase and MFO enzymes in all Ae. aegypti populations. All, except three populations, showed increased levels of ß-esterases; and all populations, except Curepe, demonstrated elevated GST levels.Metabolic detoxification of enzymes is correlated with the manifestation of DDT and PY resistance in Trinidad and Tobago populations of Ae. aegypti. The presence of this resistance also suggests that knock down (kdr)-type resistance may be involved, hence the need for further investigations. This information can contribute to the development of an insecticide resistance surveillance programme and improvement of resistance management strategies aimed at combatting the spread of dengue in Trinidad and Tobago.
Assuntos
Aedes , DDT , Inseticidas , Piretrinas , Animais , Resistência a Inseticidas , Larva , Trinidad e TobagoRESUMO
A ribosomal DNA-polymerase chain reaction (PCR) method has been developed for species identification of individuals of the five most widespread members of the Anopheles gambiae complex, a group of morphologically indistinguishable sibling mosquito species that includes the major vectors of malaria in Africa. The method, which is based on species-specific nucleotide sequences in the ribosomal DNA intergenic spacers, may be used to identify both species and interspecies hybrids, regardless of life stage, using either extracted DNA or fragments of a specimen. Intact portions of a mosquito as small as an egg or the segment of one leg may be placed directly into the PCR mixture for amplification and analysis. The method uses a cocktail of five 20-base oligonucleotides to identify An. gambiae, An. arabiensis, An. quadriannnulatus, and either An. melas in western Africa or An. melas in eastern and southern Africa.
Assuntos
Anopheles/genética , DNA Ribossômico/análise , Insetos Vetores/genética , Reação em Cadeia da Polimerase , Animais , Sequência de Bases , Primers do DNA/química , DNA Ribossômico/química , Feminino , Malária/transmissão , Masculino , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Mosquito flight tone was amplified and digitally sampled at 20,000 samples per second (Hz). Resampling of the resulting sound files at 1,000, 5,000, and 10,000 Hz allowed comparison of flight tone frequency distributions for males and females of Aedes aegypti (L.) and A. albopictus (Skuse). Frequency distributions for females of the two species did not overlap at sampling rates of 5,000 Hz or higher, whereas considerable overlap was observed at the 1,000 Hz sampling rate. Males of the two species produced flight tones higher in frequency than those of females, but similar to each other. At the highest sampling rate, seven flight tone harmonics were measured for each species. Close correspondence of the means of the flight tone harmonics (corrected for harmonic number) demonstrated that any of the harmonics may be used accurately and precisely to calculate flight tone frequency. These data indicate that flight tone differences have been selected in these species and could act as an isolating mechanism for mating.
Assuntos
Aedes/fisiologia , Voo Animal/fisiologia , Acústica , Animais , Feminino , Masculino , Comportamento Sexual Animal , Especificidade da EspécieRESUMO
Through digital sampling and resampling at 5,000 and 20,000 Hz of amplified mosquito flight sound, baseline separation was observed for flight tone frequency distributions of male and female Anopheles gambiae Giles, An. arabiensis Patton, An. merus Donitz, and An. melas Theobald. Males of the 4 species showed flight tones considerably higher than females. Up to 7 harmonics were measured for each species. Close correspondence for each individual mosquito of the means of the flight tone harmonics (corrected for harmonic number) demonstrated the accuracy and precision of the method. These data indicate that flight tone differences have been subjected to selection and may act as an isolating mechanism for mating or serve some other behavioral purpose in these mosquitoes. Individuals and swarms of sympatric species were distinguished from each other for both males and females, but the allopatric species, An. merus and An. melas, were indistinguishable.
Assuntos
Anopheles/classificação , Anopheles/fisiologia , Voo Animal/fisiologia , Animais , Feminino , Masculino , Caracteres Sexuais , Som , Especificidade da EspécieRESUMO
Isofemale lines were established containing either, both, or neither of the elevated esterase and oxidase resistance mechanisms conferring pyrethroid resistance in a Guatemalan strain of Anopheles albimanus (Wiedemann). Plots of esterase and oxidase levels for individual mosquitoes from these single families correlated with data obtained using oxidase and esterase synergists in bioassays run in the bottle format. Mixed populations of pyrethroid-resistant A. albimanus adult females were selected using DDT, permethrin, or malathion; and the esterase and oxidase levels of the individual progeny were plotted. These data showed that the 3 classes of insecticide selected the 2 mechanisms differently. These results are discussed in terms of the problem of multiresistance surveillance in the field, especially concerning pyrethroid insecticides and the interaction of agricultural and public health insecticide application.
Assuntos
Anopheles , Controle de Mosquitos , Piretrinas , Animais , Anopheles/enzimologia , DDT , Esterases/metabolismo , Feminino , Guatemala , Resistência a Inseticidas , Malation , Controle de Mosquitos/métodos , Oxirredutases/metabolismo , PermetrinaRESUMO
A simple method is described for treating 250-ml glass Wheaton bottles with insecticide, and using them as test chambers for detecting insecticide resistance in mosquito and sandfly populations. The methods for treating bottles, obtaining baseline data, and applying this technique to insects from the field are described. Sample data are presented from tests run on different vector species using a variety of insecticides. Time-mortality data from the bottle bioassay are presented alongside results from biochemical detection methods applied to the same mosquito population. The potential role, advantages, and limitations of the time-mortality bottle method are discussed.
Assuntos
Bioensaio/métodos , Culicidae , Resistência a Inseticidas , Aedes , Animais , Anopheles , Culex , InseticidasRESUMO
Optimum conditions are described for a simple, rapid, microplate-based assay that indirectly measures the differences in oxidase levels between individual susceptible, resistant, or induced mosquitoes. A small proportion (0.01-0.1) of a single mosquito is used, allowing multiple replicates of the oxidase assay. Cytochrome C is used as a positive control. The levels of oxidase found in sample populations of pyrethroid-susceptible, pyrethroid-resistant, and phenobarbital-induced Anopheles albimanus mosquitoes are characterized with the assay.
Assuntos
Anopheles/enzimologia , Resistência a Inseticidas , Peroxidase/análise , Animais , Citocromo-c Peroxidase/análise , Feminino , Oxirredutases/análise , PopulaçãoRESUMO
Anopheles pseudopunctipennis, one of the primary vectors of malaria in the southwest of Colombia, was evaluated for susceptibility to the 3 major insecticide groups (organophosphates, pyrethroids, and carbamates) by bioassay and biochemical assay. Larval populations, which were collected principally from irrigation channels in agricultural areas, where the intensity of insecticide use varied, were utilized to establish susceptibility for the 1st time in this species. The baselines for each population showed a range of biological susceptibility to the insecticides evaluated, but overall no resistance was detected according to standards established by the World Health Organization. The high sensitivity of biochemical microassays enabled the detection of a small proportion of mosquitoes with higher levels of nonspecific esterases and mixed-function oxidases from 2 areas where agricultural application of organophosphate and pyrethroid insecticides had been heavy. These differences were not sufficient to affect susceptibility as measured by bioassay. No evidence of insensitive acetylcholinesterase was observed. Absence of resistance in areas that have experienced heavy insecticide application could be explained by genetic drift, by gene flow from areas without insecticide pressure, by manner of exposure to the insecticides, or by recent changes in agricultural activities that decreased insecticide use. Baseline values were established that serve as provisional susceptibility thresholds for applying simple Centers for Disease Control and Prevention biochemical assay and bioassay methods to larvae of this anopheline species.
Assuntos
Anopheles , Inseticidas , Controle de Mosquitos , Acetilcolinesterase/análise , Animais , Anopheles/enzimologia , Bioensaio , Colômbia , Esterases/análise , Malation , Controle de Mosquitos/métodos , Oxirredutases/análise , Permetrina , Propoxur , PiretrinasRESUMO
Reduced fenitrothion susceptibility in Haitian Anopheles albimanus is documented using time/mortality measurements from otherwise standard World Health Organization (WHO) bioassays. Survival beyond a time threshold in bioassays is shown to be highly correlated with elevated non-specific esterase levels. A shift in resistance incidence from less than 20 to over 60% in a six-month period is documented using both the bioassay and microassay procedures, showing the potential of microplate assay methods in early detection of resistance. Conventional 24-hour reading of WHO bioassay data failed to detect resistance until the level reached 60%. Resistance appeared to be focal, with an increase in intensity coinciding with a fenitrothion spray cycle for malaria control.
Assuntos
Anopheles , Fenitrotion , Animais , Bioensaio , Resistência a Medicamentos , HaitiRESUMO
Little is known about the population genetics of the louse infestations of humans. We used microsatellite DNA to study 11 double infestations, that is, hosts infested with head lice and body lice simultaneously. We tested for population structure on a host, and for population structure among seven hosts that shared sleeping quarters. We also sought evidence of migration among louse populations. Our results showed that: (i) the head and body lice on these individual hosts were two genetically distinct populations; (ii) each host had their own populations of head and body lice that were genetically distinct to those on other hosts; and (iii) lice had migrated from head to head, and from body to body, but not between heads and bodies. Our results indicate that head and body lice are separate species.
Assuntos
Infestações por Piolhos , Pediculus/classificação , Pediculus/genética , Animais , DNA/análise , Cabeça , Humanos , Repetições de Microssatélites/genética , Movimento , Reação em Cadeia da PolimeraseRESUMO
Insecticide resistance is an increasing problem in vector control programmes. Until recently, the usual means of detecting it has been by bioassay, requiring the use of relatively large numbers of insects and insecticide-impregnated test papers which may be difficult to prepare and store reproducibly. William Brogdon argues for the use of biochemical microplate assays which are cheaper and easier to use, permit up to 30 assays to be made on a single insect, and give more reproducible results.
RESUMO
A microassay is described for determining protein levels in individual mosquitoes. This method requires only a small portion of a homogenate of a single mosquito. Protein levels in Anopheles albimanus are positively correlated with body weight. Protein levels for different geographic strains of Anopheles albimanus and different generations of laboratory rearings are compared.
Assuntos
Anopheles/análise , Proteínas/análise , Animais , Peso Corporal , Microquímica , Especificidade da Espécie , Espectrofotometria/métodosRESUMO
A simple microassay is described for determining protein levels in individual mosquitoes; this method should be useful on a routine basis in the field. This method requires only a small portion of a homogenate of a single mosquito. Sugar-fed adult female Anopheles albimanus showed a slow decline in protein level for 14 days post-emergence. Blood volumes and blood-meal protein levels were microassayed, and changes in protein level were monitored throughout the reproductive cycle. Blood-fed Anopheles albimanus protein levels return only slowly to pre-feeding levels following egg deposition.
Assuntos
Anopheles/análise , Proteínas/análise , Envelhecimento , Animais , Anopheles/crescimento & desenvolvimento , Sangue , Feminino , Masculino , Microquímica , Espectrofotometria/métodosRESUMO
1. A simple, rapid microassay method is described for measuring acetylcholinesterase (AChE) activity accurately and precisely in small portions of single mosquito homogenates. 2. Up to 30 microassay replicates were possible for individual insects. 3. Microassay data on individual mosquitoes were compared with conventional enzyme assay data acquired using pools of the same homogenates. 4. Under the optimum reaction conditions established, an average Vmax of 7.1 nmol/l/min/mosquito and an average Km of 1.3 x 10(-4) M were observed with acetylthiocholine iodide as substrate. 5. Variability in AChE activity within a sample population of Anopheles albimanus was observed using measurements from individual insects. 6. Such information is fundamental to comparative studies of pesticide physiology (in particular, the resistance phenomenon) in the individual mosquitoes in a population pool; this technique forms the basis for a recently developed resistance microassay.
Assuntos
Acetilcolinesterase/análise , Anopheles/metabolismo , Acetiltiocolina/metabolismo , Animais , Feminino , Hidrólise , MicroquímicaRESUMO
A straightforward method is described for determination of the activities of erythrocyte acetylcholinesterase and plasma cholinesterase from single 10-mul samples of highly diluted whole blood. The procedure is a spectrophotometric microassay adapted for use with microtitration plates. The use of an enzyme immunoassay reader facilitates the rapid analysis of large numbers of samples. An inexpensive adaptation of the method was also devised that requires no instrumentation and was evaluated under field conditions in Haiti. Either the field or laboratory versions of the method can accurately determine increments of 10% or less in the level of inhibition of acetylcholinesterase or cholinesterase in whole blood, plasma, or erythrocytes.
Assuntos
Colinesterases/sangue , Eritrócitos/enzimologia , Acetilcolinesterase/sangue , Agricultura , Haiti , Humanos , Microquímica/métodos , Espectrofotometria/métodosRESUMO
A microtiter plate spectrophotometric system has been used together with the Bradford, Ellman, and van Asperen assays to measure protein concentration (to 0.5 microgram) and the activities of acetylcholinesterase (to 10(-3) units) and carboxylesterase (to 30 micrograms beta-naphthol reaction product) in small samples such as high-performance liquid chromatographic eluate fractions. For 100-microliter samples, at least 300 Ellman acetylcholinesterase or Bradford protein assays can be conducted and read in less than 30 min, and a like number of van Asperen nonspecific esterase assays (including 1-h incubation) run in less than 90 min. The eluate from a single 20-min high-performance liquid chromatographic run of a biological sample can be collected as up to 300 fractions directly into microtiter plate wells, and the three assays run on all fractions in less than 90 min.
Assuntos
Acetilcolinesterase/análise , Hidrolases de Éster Carboxílico/análise , Acetilcolinesterase/metabolismo , Animais , Hidrolases de Éster Carboxílico/metabolismo , Cromatografia Líquida de Alta Pressão , Fígado/enzimologia , Músculos/enzimologia , Coelhos , Espectrofotometria , SuínosRESUMO
1. Optimum conditions are described for a simple, rapid microplate assay that measures glutathione s-transferase (GST) activity accurately and precisely in small portions of single mosquito homogenates. 2. Up to 10 assay replicates were possible for individual adults and larvae. Concentration of GST activity in the head/thorax region allows blood-fed mosquitoes with abdomens removed to be used in assays. 3. The method allows the use of GST activity as a biochemical character in comparative studies of populations. 4. The microplate assay detects elevated GST activities associated with DDT resistance in Anopheles arabiensis.
Assuntos
Anopheles/enzimologia , Resistência a Medicamentos , Glutationa Transferase/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Larva , Especificidade da EspécieRESUMO
Insecticide resistance has been a problem in all insect groups that serve as vectors of emerging diseases. Although mechanisms by which insecticides become less effective are similar across all vector taxa, each resistance problem is potentially unique and may involve a complex pattern of resistance foci. The main defense against resistance is close surveillance of the susceptibility of vector populations. We describe the mechanisms of insecticide resistance, as well as specific instances of resistance emergence worldwide, and discuss prospects for resistance management and priorities for detection and surveillance.
Assuntos
Controle de Insetos , Insetos Vetores , Resistência a Inseticidas , Animais , Substâncias de Crescimento , Humanos , Resistência a Inseticidas/fisiologia , Inseticidas , IvermectinaRESUMO
The reliability of a published method to predict survivorship in the WHO propoxur-resistance bioassay (WHO test) from the results of a biochemical assay for detecting the insensitivity of acetylcholinesterase (AChE) is described. For biochemical assay data from three field populations of Anopheles albimanus mosquitos, the results obtained using the method did not correlate consistently with the findings of the WHO test. A modified method is then described that eliminates the effect on the assay of factors unrelated to pesticide resistance, and it is shown that this modification can be used to predict survivorship in the WHO test for mosquitos from three study sites in Guatemala. The results show that when scored visually, the insensitive AChE microplate assay is an accurate method of estimating survival in the WHO test, regardless of whether the mosquitos tested are blood-fed or not. Recommendations are given for the application and analysis of data from the insensitive AChE microplate assay for detecting and monitoring resistance to carbamate insecticides.